The work presented aimed to elucidate the amino acid profile of a number of grapevine cultivars relevant to the South African wine industry using 738 grape must samples obtained during the 2016 and 2017 harvests. Part 1 presents the findings of the survey, while Part 2 will focus on additional information that could be extracted from the data.

Nitrogenous compounds are some of the most important yeast nutrients present in the grape juice, second only to carbon. The free amino nitrogen portion of YAN is comprised of an array of amino acids and has been reported to make up 50 – 90% of the grape must YAN. Since not all amino acids are equal in supporting the growth of the yeast, there is a preferential uptake. Accordingly, some amino acids are considered ‘good’ sources of nitrogen and others ‘poor’. Aside from ammonium, amino acids which are preferred by the yeast include glutamate, glutamine, aspartate, asparagine and arginine, whereas tryptophan, histidine, glycine and lysine are considered as poor sources of nitrogen. On the other hand, proline, the most abundant amino acid, is not considered as a source of YAN during fermentative conditions.

The oenological relevance of amino acids also resides in the formation of higher alcohols and esters due to the presence of branched-chain (valine, leucine and isoleucine) and aromatic (tryptophan, tyrosine and phenylalanine) amino acids. These amino acids are the precursor molecules for certain aroma compounds. Moreover, it was found that the amino acid composition influences the concentration of other compounds for which amino acids are not the direct precursors. Examples of these include ethanol, acetic acid, as well as fatty acids.

Due to the central role of nitrogenous compounds in yeast metabolism and, consequently, the modulation of the organoleptic qualities of the resulting wine, knowledge of the amino acid profile is advantageous. Work done for the past seasons at the Department of Viticulture and Oenology (DVO) focused on the elucidation of the amino acid profile of a number of grapevine cultivars relevant to the South African wine industry.

Samples

The amino acid profile of 738 commercial grape juices was obtained over the 2016 and 2017 harvests. Samples were collected from various grape-growing districts across the Western Cape region of South Africa. The survey followed an unsupervised format, resulting in the collection of 13 different cultivars, seven white and six red. The cultivars collected included: Cabernet franc (n=13), Cabernet Sauvignon (n=38), Chardonnay (n=97), Chenin blanc (n=176), Cinsaut (n=15), Grenache blanc (n=17), Merlot (n=29), Pinotage (n=12), Roussanne (n=15), Sauvignon blanc (n=219), Sémillon (n=16), Shiraz (n=51), and Viognier (n=40). All cultivars were harvested at a ripeness level suitable for commercial winemaking, according to the cellars participating in the survey. The determination of individual amino acids was done using the AccQ-Tag Ultra amino acid kit (Waters) and High Pressure Liquid Chromatography, followed by UV-Vis detection.

Proline and arginine

Proline and arginine were found to be the most abundant amino acids, with an average of 697.7 mg/L for proline (range 33.2 – 3 445.4 mg/L) and 388.4 mg/L for arginine (range 13.6 – 1 616.6 mg/L) across all vintages, regions and cultivars (Figure 1). Proline accumulation in the grape berry occurs towards the end of ripening, typically in the last four to six weeks before harvest. The reason for proline accumulation in plants is, however, a topic of debate. Proline accumulation is considered a stress-response mechanism, protecting tissues against oxidative and osmotic stress.

Merlot, Cabernet Sauvignon and Chardonnay were found to be the highest proline accumulators (proline to arginine ratio >1), whereas Cinsaut, Pinotage and Grenache blanc were found to be the lowest proline accumulators. Sauvignon blanc on the other hand was observed to have, on average, equal concentrations of proline and arginine. Proportionally, proline was observed to make up from 14.4% (Grenache blanc) to 69.21% (Merlot), on average 35.7% of the grape juice amino acid content.

FIGURE 1. Examples of compound concentration and distribution per cultivar for the samples included in the survey for the two most abundant amino acids. Concentration is expressed in mg/L.

Abundant amino acids

Other than proline and arginine, on average, glutamine (average 111.6 mg/L, range 61.1 – 216.4 mg/L), tryptophan (105.7 mg/L, 47.3 – 214.3 mg/L), γ-amino butyric acid (GABA, 100.2 mg/L, 69.2 – 136.6 mg/L), and alanine (85.2 mg/L, 38.6 – 145.1 mg/L) were found to be the most abundant amino acids. Aromatic amino acids were observed to make up a larger proportion of the total amino content for each cultivar, on average 7.1% of the amino acid content compared to 3.5% for the branched-chain amino acids. Proportionally, Cabernet Sauvignon and Merlot had the lowest aromatic amino acid content, with only 4.1% and 4.2%, respectively. These cultivars were also observed to have amongst the lowest proportions of branched-chain amino acids (Cabernet Sauvignon 2.8% and Merlot 2.6%), along with Chardonnay 2.6%. Moreover, Roussanne was found to have, on average, the highest aromatic amino acid content, in both absolute terms (264 mg/L) and proportionally (13.4%).

In terms of the branched-chain amino acids, Cinsaut and Roussanne were found to have the highest proportions, with 4.6% and 4.5% being found, respectively. Therefore, as Roussanne contains high concentrations of these precursor molecules (both aromatic and branched-chain amino acids), it can be identified as a cultivar with a lot of aromatic potential in terms of the production of fusel alcohols and esters. However, these positive aroma compounds are only produced when the total YAN concentration is capable of fulfilling the full biosynthetic requirement of the yeast. As Roussanne has been identified as a cultivar which has a very low total YAN content (average 132 ± 34 mg N/L, Petrovic et al., 2019), this cultivar will most likely require nutrient supplementation in the form of DAP or complex nutrients to realise its full aromatic potential.

Least abundant amino acids

Ornithine (average 2 mg/L, range 0.4 – 4.8 mg/L), glycine (3.3 mg/L, 1.1 – 5.3 mg/L), methionine (3.6 mg/L, 0.7 – 9.9 mg/L), and lysine (3.9 mg/L, 2.3 – 6.1 mg/L) were found to have the lowest concentrations, both in terms of the overall average, as well as per cultivar. The low concentration of ornithine is most likely due to its central role in nitrogen metabolism, acting as a precursor molecule for the formation of the most abundant amino acids, arginine and proline. As Saccharomyces cerevisiae, the most widely yeast used for fermentation, is not able to efficiently metabolise glycine and lysine, these amino acids are considered as a poor source of nitrogen for this yeast. However, these amino acids may be assimilated by some non-Saccharomyces yeasts.

Take home message

Combining the knowledge of YAN levels with the amino acid profile can be useful when deciding on nutrient additions. For example, Merlot and Cabernet Sauvignon were found to have the lowest proportions of aromatic and branched-chain amino acids, with Roussanne having the highest proportion of these precursors of fruity and floral aromas. As Cabernet Sauvignon and Merlot have been found to have very low total YAN concentrations, these cultivars would in most cases require nitrogen supplementation to ensure the completion of fermentation. However, the addition of complex nutrients (which may contain varying concentrations of these branched-chain and aromatic amino acids) may be a more beneficial supplementation strategy for these cultivars compared to ammonia addition in the form of diammonium phosphate (DAP). On the other hand, as Roussanne already has high concentrations of these precursor molecules, the addition of (more cost-effective) DAP may be sufficient to ensure not only the completion of fermentation, but to ensure the formation of favourable organoleptic qualities in the final wine.

Abstract

This type of composition profiling is especially relevant in terms of identifying and meeting nitrogen demands of various yeast strains, Saccharomyces and non-Saccharomyces. This is the first time such extensive work focused on amino acid composition was undertaken.